Crystal Structure of Escherichia coli PdxA, an Enzyme Involved in the Pyridoxal Phosphate Biosynthesis Pathway

Abstract

Pyridoxal 5-phosphate is an essential cofactor for many enzymes responsible for the metabolic conversions of amino acids. Two pathways for its de novo synthesis are known. The pathway utilized by Escherichia coli consists of six enzymatic steps catalyzed by six different enzymes. The fourth step is catalyzed by 4-hydroxythreonine-4-phosphate dehydrogenase (PdxA, E.C. 1.1.1.262), which converts 4-hydroxy-L-threonine phosphate (HTP) to 3-amino-2-oxopropyl phosphate. This divalent metal ion-dependent enzyme has a strict requirement for the phosphate ester form of the substrate HTP, but can utilize either NADP ؉ or NAD ؉ as redox cofactor. We report the crystal structure of E. coli PdxA and its complex with HTP and Zn 2؉. The protein forms tightly bound dimers. Each monomer has an ␣/␤/␣-fold and can be divided into two subdomains. The active site is located at the dimer interface, within a cleft between the two subdomains and involves residues from both monomers. A Zn 2؉ ion is bound within each active site, coordinated by three conserved histidine residues from both monomers. In addition two conserved amino acids, Asp 247 and Asp 267, play a role in maintaining integrity of the active site. The substrate is anchored to the enzyme by the interactions of its phospho group and by coordination of the amino and hydroxyl groups by the Zn 2؉ ion. PdxA is structurally similar to, but limited in sequence similarity with isocitrate dehydrogenase and isopropylmalate dehydrogenase. These structural similarities and the comparison with a NADPbound isocitrate dehydrogenase suggest that the cofactor binding mode of PdxA is very similar to that of the other two enzymes and that PdxA catalyzes a stepwise oxidative decarboxylation of the substrate HTP.Pyridoxal 5Ј-phosphate, the catalytically active form of vitamin B 6 , is an important cofactor for many enzymes responsible for the metabolic conversions of amino acids. Vitamin B 6 (pyridoxine) and its derivatives are also efficient singlet oxygen quenchers and potent fungal antioxidants (1). Two different pathways for de novo synthesis of pyridoxine are now recognized. One of these, found in all Archaea, eukaryotes, and in some bacteria, uses the singlet oxygen resistance (SOR1(Pdx1)) gene product, a highly conserved enzyme (2). A number of eubacteria, including Escherichia coli, utilize a specific pathway for pyridoxal phosphate synthesis that is distinct and has been characterized for some time (3). Analysis of sequences from several genomes has revealed that organisms may encode either the SOR1 or E. coli-like pyridoxine biosynthesis genes, but not both (4).The pyridoxal 5Ј-phosphate biosynthesis pathway has been well characterized in E. coli, and consists of six enzymatic steps, beginning with erythrose 4-phosphate and deoxyxylulose 5-phosphate (5-7). All of the enzymes in this pathway have been identified. Of the six enzymes, PdxA, 1 erythromate-4-phosphate dehydrogenase (PdxB), and pyridoxine-5Ј-phosphate oxidase are unique to this pathway (5), whereas SerC and GapA also funct...

Structural Insights into the Mechanism of the PLP Synthase Holoenzyme from Thermotoga maritima,

“…YaaE is an α/β three layer sandwich containing twelve β-strands, six α-helices and three 3 10 helices ( Figure 1c) and is characteristic of class I amidotransferases. The structure of YaaE in complex with YaaD is similar to the structures of YaaE from B. subtilis (17), YaaE from B. stearothermophilus (PDB ID 1Q7R) and Pdx2 from P. falciparum (26).…”

“…Helix α0 is involved in extensive interactions with YaaE and helps form one side of the proposed ammonia channel. Comparing YaaD to PdxS, a series of smaller conformational changes in the 3 10 -helix (237-240), the β6-α6 loop, α8 and α8a connect adjacent active sites within the PLP synthase complex. In the structure of YaaD residues 46-56 form an active site loop.…”

“…There are two distinct PLP biosynthetic pathways that have not yet been found to coexist in the same organism (5). The Escherichia coli pathway has been extensively studied (6)(7)(8)(9)(10)(11)(12). In this pathway, PdxJ catalyzes the formation of pyridoxine 5'-phosphate (3, PNP) from 3-phosphohydroxy-1-aminoacetone 2 and 1-deoxy-D-xyulose 5-phosphate 1.…”

Crystal Structure of the PdxY Protein from Escherichia coli

“…In many bacteria and plants, PLP is synthesized by a de novo pathway, but most cells rely on a nutritional source of vitamin B 6 , i.e., pyridoxine (PN), pyridoxal (PL), or pyridoxamine (PM) (18). All cells, however, have a salvage pathway for reutilizing the PLP that is liberated during protein turnover (21).…”

Assignment of function to a domain of unknown function: DUF1537 is a new kinase family in catabolic pathways for acid sugars

“…The SSN for the PdxA family filtered with an alignment score of 80 (∼47% sequence identity) revealed that the members that are encoded genome proximal to DUF1537 proteins (designated a PdxA2 subgroup) are separated from the proteins (PdxA) that catalyze the NAD(P) + -dependent oxidative decarboxylation of 4-hydroxy-L-threonine 4-phosphate (4HT-4P) to form 3-amino-1-hydroxyacetone 1-phosphate in the biosynthetic pathway for pyridoxal 5′-phosphate (PLP) (SI Appendix, Fig. S5) (26,27), suggesting a distinct function.…”